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Nucleotide production process

a production process and nucleotide technology, applied in biochemistry apparatus and processes, fermentation, enzymes, etc., can solve the problems of difficult separation, difficult crystallisation, and methods for producing nucleotides, and achieve high yield, high purity, and low solubility in water

Inactive Publication Date: 2017-05-11
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention solves the problem of producing high-purity nucleotides by using a double-enzyme catalysis method, which results in a whole biocatalytic production of nucleotides from yeast RNA. The combination of ion exchange resin separation technology and solvent crystallization method purifies the nucleotides to a high purity, with a total recovery from RNA to high-purity nucleotides of more than 85%, and a purity of more than 99.9%. The process is simple, environmentally-friendly and has low costs. The present invention also simplifies the post-purification process, reduces the using amount of enzymes, acids, and bases, and improves the recovery and purity of products. This solution meets the demand for nucleotides of high-purity in the infant food and pharmaceutical industries.

Problems solved by technology

However, as there exsit some chemical toxic agents and many chemical byproducts, which are difficult to be completely removed from products, as well as some factors such as high requirements on equipments which are inappropriate to industrial productivity, the chemical synthesis method has been eliminated; the natural raw materials extracting method is difficult to meet the needs of the society due to limited sources of raw materials and being unable to realize a large mass production; it is feasible to use cheap raw material to produce nucleotides if the microbiological fermentation method is employed, however, this method is restricted as the ability of accumulating nucleotides for microbes is limited, the products are different to across through the cell membrane and the industrialisation is restricted due to high equipment assets, low concentration of products and high separation costs; the fermentation-catalyzed coupling method is a modified method aiming to overcome the shortcoming that nucleotides are different to across through the cell membrane, in this method, nucleosides were firstly produced by fermentation method, and then were phosphorylated to nucleotides.
However, as there exsit byproducts such as 2′-phosphate, 3′-phosphate or diphosphate during the process of phosphorylation, which making separation difficult.
Patent application (CN1086219A) discloses a method for preparing nucleotides by chemically phosphorylating nucleosides, in which lower alkyl esters of phosphoric acid are used as the solvent and trihalo-phosphorus oxides are used as phosphorylating agents, however, there appear a large number of byproducts during the production process, which do not meet the requirements for food safety, meanwhile, acetic acid / ammonium acetate buffer is used during the elution process of resins, which bring difficulties for crystallisation.
In summary, the existing methods for producing nucleotides have some defects such as complexed production process, higher cost, being hard to separate, lower safety and purity, and being difficult to realize the industrial production and so on.
The nucleotides, as biological products, have high nutrition and high medicinal value, which are commonly used for infant food and pharmaceutical industries, but the requirements of high purity and high quality make the existing process for producing nucleotides be difficult to meet the demands of infant food and pharmaceutical industries.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Nuclease P1

[0075]A seed culture medium and a fermentation medium containing the following ingredients: 50 g / L glucose, 5 g / L peptone, 0.5 g / L KH2PO4, 0.5 g / L K2HPO4.3H2O, 0.4 g / L CaCl2 and 0.4 g / L ZnSO4 were prepared respectively. The pH was adjusted to 6.5 before sterilization.

[0076]Preparation of nuclease P1: Penicillium citrinum N409 was inoculated in the seed culture medium prepared above, cultured at 28° C., 200 rpm for 18 hours, and then inoculated into the fermentation medium with an inoculation quantity of 10% (v / v), cultured at 28° C., 200 rpm for 24 hours, and then the fermentation broth was centrifuged at 8000 rpm, 4° C. for 15 minutes, the supernatant was taken to obtain 420 U / ml of nuclease P1 solution, which was refrigerated at 4° C. until used.

[0077]All nuclease P1 used in the following examples 2-9 was prepared according to the above method.

example 2

Preparation of Nucleotides

[0078]36 g RNA of yeasts was weighed, and formulated into a 6% nucleic acid solution (in which NaOH was added to help dissolving before adjusting the pH to 5.5 with HCl), warmed to 70° C., 21000 U nuclease P1 solution preheated at 45° C. (about 50 ml) was added thereinto, and the reaction is performed for 4.5 hours at 70° C., and then 0.9 g activated carbon was added, the enzymolysis was held for 40 minutes, and then centrifugation, suction filtration and ultra-filtration were performed to obtain 610 ml mixture solution of nucleotides. After detection, the enzymolysis ratio of nucleotides was 85.54%, and the concentration of each nucleotide in the mixture solution of nucleotides was UMP 10.56 g / L, GMP 16.83 g / L, CMP 10.87g / L and AMP 13.5293 g / L.

[0079]50 g pretreated cation exchange resin Amberlite IR-120 and anion exchange resin Amberlite IRA-68 were weighed respectively and packed into columns. The mixture solution of nucleotides obtained above was loaded ...

example 3

Preparation of Nucleotides

[0082]50 g RNA of yeasts was weighed, and formulated into a 5% nucleic acid solution (in which NaOH was added to help dissolving before adjusting the pH to 5.5 with Ha), warmed to 50° C., and then 100000 U nuclease P1 solution preheated at 45° C. (about 240 ml) and 40 U adenosine deaminase powder (about 40 mg) were added thereinto, then the enzymatic hydrolysis-deamination reaction was performed at 50° C., during which 3 mol / L HCl was added to control the pH value at 5.5±0.2, after 5 hours, 2 g activated carbon was added, the enzymolysis was held for 30 minutes, and then centrifugation, suction filtration and ultra-filtration were performed to obtain 1020 ml mixture solution of nucleotides. After detection, the enzymolysis ratio of nucleotides was 88.56%, the converting rate of AMP was 99.9999% and the concentration of each nucleotide in the mixture solution of nucleotides was UMP 8.7692 g / L, GMP 13.9790 g / L, CMP 9.0296 g / L and IMP 11.6635 g / L.

[0083]100 g p...

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Abstract

A nucleotide production process comprises: decomposing an RNA by using a nuclease P1 so as to obtain nucleotides AMP, GMP, CMP and UMP, converting part or all of the nucleotide AMP into a nucleotide IMP by using adenosine deaminase, separating the obtained nucleotide by using an ion exchange resin, and then performing concentration and crystallization to obtain purified nucleotides AMP, GMP, CMP, UMP and IMP or obtain purified nucleotides GMP, CMP, UMP and IMP. The whole biocatalysis production of nucleotides is implemented by using a double-enzyme catalysis method, and high-purity nucleotides are obtained by using an ion resin separation technology and a solvent crystallization method; and the production process is simple and environmentally-friendly, and has low costs, high product safety and purity.

Description

TECHNICAL FIELD[0001]The present invention belongs to the technical field of biosynthesis and separation, in particular relates to a nucleotide production process.BACKGROUND ART[0002]Nucleotide is a compound consisting of three materials comprising a base (purine base or pyrimidine base), a ribose (ribose or deoxyribose) and a phosphoric acid, which is the structural unit of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Depending on the kinds of bases, nucleotides can be divided into adenylate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), thymidylate (TMP) and inosine monophosphate (IMP) and the like. Nucleotide compounds have important biological functions, they involve almost all biochemical reactions in vivo, including the following aspects: (1) existing as the structural units of biomolecules ribonucleic acid (RNA) and deoxyribonucleic acid (DNA); (2) adenosine triphosphate (ATP) in the cell acts as an energy currency, ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/30
CPCC12P19/30C12Y301/11005
Inventor YING, HANJIECAO, ZHICHEN, YONGCHEN, XIAOCHUNWU, JINGLANXIE, JINGJING
Owner NANJING UNIV OF TECH
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